The techniques for identifying RNA or DNA sequences (target sequences) in a sample involve hybridizing the target sequence with a labelled probe and detecting the duplex which has been formed, after the unhybridized probe has been removed.
These techniques are commonly used, in particular, for diagnosing viral or infectious diseases, for medical or genetic research, for identifying clones, for analysing transcribed genes, etc.
Two types of probe are employed, i.e. so-called long probes (generally of a size larger than 100 bases) and short probes, whose sizes vary between 10 and 30 bases in length. The probes which are most commonly used in medical diagnosis are synthetic oligonucleotides which are coupled to a label, either by direct attachment to this label or by coupling to a ligand; in this latter case, detection is effected by way of a label which is fixed to this ligand.
Radioactive or cold labels can be used.
A large number of labelling techniques are described, for example by SAMBROOK et al., [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)]. In the case of radioactive labels, these methods include, for example, labelling synthetic oligonucleotides 5' with .gamma..sup.32 P-ATP using the enzyme polynucleotide kinase, or else incorporating radioactive dCTP or dATP while labelling by nick translation.
Biotin may be mentioned, in particular, as an example of a non-radioactive label which can be incorporated chemically into synthetic oligonucleotides (Application PCT WO 89/12462); biotinylated analogues of UTP can also be incorporated into double-stranded DNAs by nick translation.
In general, biotin is detected by being bound to avidin, which is itself used as a support for attaching fluorescent molecules, enzymes or other detectable compounds.
In other non-radioactive labelling systems, haptens (dinitrophenyl group, digoxigenin) replace biotin and specific labelled antibodies are used as indicators in the system.
The sensitivity of molecular hybridization assays is an important factor which is often limiting when applying diagnostic tests in molecular biology.
The sensitivity of an assay depends directly on the labelling which is used to visualize the hybridized probe. One of the ways of improving the sensitivity of an assay is to amplify the signal by increasing the number of labels which are incorporated into the probe in order to obtain so-called "polylabelled" probes.
With this aim in mind, it has been proposed, for example, that the enzyme terminal transferase should be used, which enzyme is able to extend a single-stranded DNA chain by adding nucleotide analogues, for example biotinylated nucleotides, to its 3' end. The main drawback of this method is that the enzyme adds an arbitrary number of nucleotides to the end of the single-stranded DNA chain. This results in a mixture of products of different lengths which in turn makes the labelling difficult to reproduce and produces heterogeneous probes which are difficult to standardize and therefore unsuitable for medical diagnosis.
COLLINS (Application EP 204 510) proposes a variant in which the enzyme terminal transferase is used to extend the probe by adding a homopolymeric (poly A) tail to it. The label then consists of a homopolymer (poly T) into which detectable molecules are incorporated. However, this system does not completely eliminate the drawbacks which result from the impossibility of controlling the action of the enzyme.
It has also been proposed to amplify the signal by using polylabelled oligonucleotide probes which are branched or else connected to each other by polynucleotide "bridges" which hybridize to their ends, as described in Applications EP 292 128 and EP 450 594, respectively, which are in the name of SEGEV.
U.S. Pat. No. 4,882,269 describes a system in which polylabelled secondary probes hybridize to several sites on a primary probe.
Application EP 0 317 077 and also Application PCT WO 92/02526 describe a system in which the secondary probes consist of polylabelled polynucleotide constructs in comb form.
These systems suffer from the drawback of requiring the synthesis of a large number of different oligonucleotides and the implementation of hybridization and/or ligation steps in order to form the final constructs. In addition, it is difficult to obtain final products having uniform characteristics and it is awkward to purify these products.
In particular, if there is a need to detect several different target sequences, it is then necessary, for each of these sequences, either to synthesize a new probe or to use an adapter, which is an oligonucleotide which hybridizes with the target sequence to be detected, on the one hand, and with the probe, on the other hand.